It uses red blood cells or neutral synthetic materials (for example, latex particles), on the surface of which antigens (bacterial, viral, tissue) or antibodies are sorbed. Their agglutination occurs when appropriate sera or antigens are added. Red blood cells sensitized with antigens are called antigenic erythrocyte diagnosticum and are used to detect and titrate antibodies. Erythrocytes sensitized with antibodies. are called immunoglobulin erythrocyte diagnosticums and are used to detect antigens.

The passive hemagglutination reaction is used to diagnose diseases caused by bacteria (typhoid and paratyphoid fever, dysentery, brucellosis, plague, cholera, etc.), protozoa (malaria) and viruses (influenza, adenoviral infections, viral hepatitis B, measles, tick-borne encephalitis, Crimean hemorrhagic fever, etc.), as well as to determine certain hormones, identify the patient’s increased sensitivity to medicines and hormones such as penicillin and insulin.

Passive hemagglutination reaction (RPHA). The passive hemagglutination test is a sensitive method of serological diagnosis and is used for both early and retrospective diagnosis, as well as to determine the immunopogic state of vaccinated individuals. In patients with tularemia, antibodies are usually detected at the end of the 1st or 2nd week of the disease; after 1-1.5 months, RPHA titers reach maximum levels (1: 100,000-1: 20,000, less often higher), after which they decrease to the level 1:100-1:200 are stored for a long time.

In vaccinated people, antibodies are also constantly detected, however, in lower titers, not exceeding 1:2000-1:5000 1-1.5 months after vaccination, and remain for several years at a low level of 1:20-1:80.

The antigen for staging RPHA is tularemia erythrocyte diagnosticum (antigenic). The drug is formalinized sheep red blood cells, sensitized with tularemia antigen, available in liquid and dry form. Liquid preparation - a 10% suspension of red blood cells in a formaldehyde solution of 10% concentration. Dry lyophilized preparation is a vacuum-dried 10% suspension of red blood cells without a preservative. Before use, it is diluted according to the directions on the label. To set up the reaction in polystyrene plates, both drugs are used in a 2.5% concentration, and when setting up the reaction in microvolumes - at a 0.5% concentration.

Technique for setting up RPGA. The test sera are diluted with physiological solution 1:5 (1:10) and heated at 56 degrees C for 30 minutes. After this, in order to remove heterogeneous antibodies to sheep erythrocytes, the sera are treated with a 50% suspension of formalinized sheep erythrocytes. To do this, add red blood cells at the rate of 2 drops (0.05 ml) per 1 ml of serum and mix thoroughly by shaking. The serum is left until the erythrocytes have completely settled, or is centrifuged after one hour at room temperature, after which it is ready for examination.

The dilution liquid is poured in a volume of 0.5 ml into a row of wells on a polystyrene plate. During the preliminary study of sera, it is advisable to test them by setting up the reaction in a short row of the plate (6-wells). If antibodies are detected in a short series, the sera are retested in a long series of dilutions (12 wells). After spilling the dilution liquid, add 0.5 ml of test sera in a 1:5 dilution to the first well of each row (short or long). Then the same volumes of serum are titrated with twofold dilutions. Thus, serum dilutions are obtained in the short series from 1:10 to 1:320, and in the long series from 1:10 to 1:20480. After titration of the sera, one drop (0.05 ml) of a working 2.5% suspension of sensitized erythrocytes is added to each well. The contents of the plates are thoroughly shaken until a homogeneous suspension is obtained. The plates are left at room temperature on a stationary table surface. Preliminary recording of the reaction is carried out after 2-3 hours, the final determination of the titer is made after complete sedimentation of red blood cells in the wells. The following controls are provided for the reaction: 1) test serum diluted 1:10 in a volume of 0.5 ml + 1 drop of a 2.5% suspension of unsensitized erythrocytes; 2) dilution liquid in a volume of 0.5 ml + 1 drop of a 2.5% suspension of unsensitized erythrocytes; 3) dilution liquid in a volume of 0.5 ml + 1 drop of a 2.5% suspension of sensitized erythrocytes. All controls should give a clearly negative reaction.

Accounting and assessment of RPGA. The reaction is assessed according to the following scheme:

1) sharply positive reaction(++++) - red blood cells fall to the bottom of the hole in an even layer in the form of an “umbrella”, which often has a scalloped melting of the edges;

2) positive reaction (+++) - red blood cells cover at least 2/3 of the bottom of the well;

3) weakly positive reaction (++) - the agglutinate is small and located in the very center of the well;

4) questionable reaction (+) - around the sediment of erythrocytes in the very center of the well there are individual grains of agglutinate;

5) negative (-) - at the bottom of the hole, red blood cells settle in the form of a “button” or a small ring with smooth, sharply defined edges.

The serum titer is taken into account based on the last dilution of the serum, which gave a very clear reaction (at least three pluses). A dilution of 1:100 or higher is considered a diagnostic titer; however, just as in the case of RA, it is necessary to monitor its increase.

TPHA in tularemia is quite specific and detects some cross-reactions only with brucellosis sera. Differential diagnosis is possible by the height of titers in TPHA, which are much higher with a homologous antigen.

Technique for setting up RPHA in microvolumes. RPHA can be performed in microvolumes using a Takachi-type microtiter (or round bottom microtiter plates with micropipettes), which allows material to be titrated in volumes of 25 µl and 50 µl. The technique of setting up reactions, the sequence of all operations is the same as in the study in polystyrene plates. However, it should be borne in mind that the sensitivity of the micromethod is usually one dilution (ie, 2 times) lower than that of the macromethod.

To set up the reaction in a microtiter using a dropper pipette, a dilution liquid in a volume of 50 μl is introduced into each well. Then, using titrators with a 50 μl head, the test serum is taken by immersing the head into it. Make sure that the liquid has filled the titrator head. The titrator with serum is transferred to the first well and, holding it in a vertical position, make several rotational movements in both directions. Then the titrator is transferred to the next well and the manipulation is repeated. Titration can be carried out simultaneously in several rows. After titration of the entire row, the titrator is washed with distilled water (with a change of 2 portions) by means of rotational movements, water is removed from the head with a swab and burned on a burner flame.

After titration, add 25 μl of erythrocyte diagnostic fluid to the wells. The concentration of the diagnosticum for RPHA in microvolumes should be 0.5% (i.e., a 2.5% suspension of erythrocytes is additionally diluted 5 times). After addition of erythrocytes, the plates should be shaken gently until a homogeneous suspension is obtained. Results can be recorded already after 1-1.5 hours, which is a significant advantage of RPHA in a microtiter. In addition, this method requires a small amount of all the ingredients of the reaction and the test sera.

The reaction is taken into account according to the following scheme:

1) "+" - complete hemagglutination, in which erythrocytes fall to the bottom of the well in a uniform layer in the form of an "umbrella", occupying at least 2/3 of the bottom;

2) "+ -" - partial hemagglutination, in which erythrocytes fall to the bottom in the form of a loose ring of a small size;

3) "-" - the absence of hemagglutination, when the erythrocytes fall to the bottom in the form of a small button or ring with a smooth edge.

The specificity of a positive result obtained in RPHA can be tested using a three-component reaction - the passive hemagglutination inhibition reaction (PHA).

Technique for setting up RTPGA. This reaction is used to confirm the specificity of a positive RPGA result when it is in doubt or is of particular epidemiological interest. The mechanism of the reaction is the specific inhibition of hemagglutination when a suspension of killed tularemia bacteria is added to the test serum. Three components interact in the reaction: test serum, specific tularemia antigen and antigenic erythrocyte diagnosticum RTPHA is usually placed in a row of 7-8 wells. It is advisable to install a repeated RPGA in parallel with the RTPGA. 0.25 ml of dilution liquid is poured into two rows of wells, then the test serum in a volume of 0.25 ml is added to the first wells of both rows and titration is performed. Two identical rows of serum dilutions are obtained. Add 0.25 ml of dilution liquid to all wells of the second row, and 0.25 ml of a suspension of tularemia bacteria to the wells of the first row. Tularemia diagnosticum is used (containing 25 billion tularemia bacteria in 1 ml), previously diluted 50 times. This suspension contains 500 million bacteria in 1 ml or 125 million in a volume of 0.25 ml. After adding the antigen, the plate is left for 1 hour at room temperature, after which one drop (0.05 ml) of erythrocyte diagnosticum is added to all wells of both rows, the plate is shaken and left on a flat table surface. Accounting is carried out after 2-3 hours.

Accounting and assessment of RTPGA. If the test serum contains specific tularemia antibodies, they are neutralized by the added antigen and hemagglutination will not occur in the first row of wells, or, with a high serum titer, hemagglutination will be observed in a smaller (2-4) number of wells than in the row with RPHA. In this case, the specificity of the results was confirmed. If hemagglutination is noted in both rows, i.e. If the results of RTPGA and RPGA coincide, this indicates the absence of tularemia antibodies in the test serum. In this case, the primary result of RPGA is considered nonspecific.

Technique for staging RTHG in microvolumes. RTPGA, like RPGA, can be performed in microvolumes using a Takachi-type microtiter. To do this, add 0.25 μl of diluting liquid into the wells of microplates in two rows of 7-8 wells each. Then, using a titrator, 0.25 μl of the test serum is added and titrated in both rows. After this, 25 μl of tularemia antigen (the concentration of which is 500 million tularemia bacteria in 1 ml) is added to each well in the first row, and 25 μl of dilution liquid is added to the second row. The plates are left for 1 hour at room temperature, after which 25 μl of antigenic zritrocytic diagnosticum (0.5% concentration) is added to all wells of both rows. Accounting and evaluation of the results are carried out similarly to reactions in macrovolumes.

Reaction indirect hemagglutination(RIGA).

RIGA is used in two versions: with known antibodies to detect antibodies with known antibodies to detect antibodies. This reaction is specific, and it is used to diagnose diseases caused by bacteria and rickettsia. To carry out RIGA, erythrocyte diagnostics are used, prepared by adsorption of AG or AT on erythrocytes, depending on the purpose of the study (Fig. 10.3). In positive cases, the degree of erythrocyte agglutination is marked with pluses. Four pluses evaluate a reaction that has the form of a thin film of adhesive red blood cells (umbrella) covering the bottom of the test tube; the presence of a film with scalloped lace edges is indicated by two pluses. The titer is taken to be the maximum dilution of the test material that caused agglutination of erythrocytes by two pluses.

Rice. 10.3.

1 - red blood cells, 2 - erythrocyte AG, 3 - conjugated AG, 4-AT

RHA and hemagglutination inhibition reaction (HAI).

As already noted, RHA is based on the ability of erythrocytes to stick together when certain antigens are adsorbed on them. Allantoic and amniotic fluid, a suspension of chorion-allantoic membranes of chicken embryos, suspensions and extracts from cultures or organs of animals infected with viruses, and native infectious material are used as test material for hemagglutination. RGA is not a serological reaction, since it occurs without the participation of immune serum and is used to select the working dilution of an antigen for staging an RGA or the presence of an antigen (virus) in the test material (for example, for influenza). The reaction uses red blood cells from animals, birds, and humans with blood group I (0). To set up an approximate RGA, a drop of a 5% suspension of erythrocytes and a drop of the test material are applied to a glass slide and mixed thoroughly. At positive result after 1-2 minutes, the appearance of flocculent agglutination of erythrocytes is observed macroscopically. To set up RGA in an unfolded row, doubly increasing dilutions of the test material are prepared in physiological solution in a volume of 0.5 ml in the wells of polystyrene plates. 0.5 ml of a 0.25-1% suspension of red blood cells is added to all test tubes. The results are taken into account after complete sedimentation of erythrocytes in the control (red blood cells + saline solution). The reaction is taken into account by the nature of the erythrocyte sediment. In positive cases, the degree of agglutination is marked with pluses. A reaction that looks like a thin film of sticky red blood cells covering the bottom of a test tube (umbrella) is assessed with four pluses; a reaction with gaps in the film is marked with three pluses; the presence of a film with scalloped lacy edges of sticky red blood cells is marked with two pluses; a flocculent sediment of red blood cells surrounded by a zone of agglutinated lumps erythrocytes, corresponds to one plus. A sharply defined erythrocyte sediment, indistinguishable from the control, indicates the absence of agglutination. The titer is taken to be the maximum dilution of the test material that caused agglutination of erythrocytes by two pluses. If the RGA result is positive, the study is continued, determining the type of isolated virus using RT GA with type-specific sera. RTGA is based on the property of antiserum to suppress viral hemagglutination, since the virus neutralized by specific antibodies loses the ability to agglutinate red blood cells. For tentative typing of viruses, the droplet method on glass is used. To definitively establish the type of the isolated virus and titrate the antibodies in the sera, an expanded X-ray test is placed in test tubes or wells. For this purpose, double dilutions of sera are prepared in physiological solution and dispensed into 0.25 ml. To the serum dilutions add one drop of material containing the virus and one drop of a 1% suspension of red blood cells. When using RTGA to determine the type of virus, type-specific sera are used, which are added to an equal volume of the working dilution of AG. The type of the isolated virus is determined by the specific immune serum that showed the highest antibody titer to this virus. RGA and RTGA are widely used for diagnostics viral infections(tick-borne encephalitis, influenza, etc.) in order to detect specific antibodies and to identify many viruses by their antibodies.

11621 0

Serological reactions are designated in accordance with the phenomena accompanying the formation of the antigen-antibody complex during the interaction of components with different properties. There are reactions of agglutination, precipitation and lysis.

Agglutination reaction (RA)

The agglutination reaction (RA) is based on the use of a corpuscular antigen (a suspension of bacteria, sensitized erythrocytes, latex particles, etc.) interacting with specific antibodies, as a result of which the resulting antigen-antibody complex precipitates. This reaction is widely used in laboratory practice for the serological diagnosis of bacterial infections and for the identification of isolated microorganisms.

RA is used to diagnose many infectious diseases: brucellosis (Wright, Heddleson reaction), tularemia, leptospirosis (RAL - Leptospira agglutination and lysis reaction), listeriosis, typhus (RAR - Rickettsia agglutination reaction), shigellosis, yersiniosis, pseudotuberculosis, etc.

Indirect or passive agglutination reaction (RIGA or RPGA).

To stage this reaction, red blood cells of animals (sheep, monkey, guinea pigs, some birds) sensitized with antibodies or antigen are used, which is achieved by incubating a suspension of red blood cells and a solution of antigen or immune serum.

Diagnosticums obtained on the basis of erythrocytes sensitized with antigens are called antigen erythrocyte diagnosticums. They are intended for the determination of antibodies in serial dilutions of blood sera, for example, erythrocyte shigella diagnosticums, erythrocyte salmonella O-diagnosticums.

Accordingly, diagnostics based on erythrocytes sensitized with specific immunoglobulins are called antibodies(immunoglobulin) diagnosticums and they serve to detect antigens in various materials, for example, erythrocyte immunoglobulin diphtheria diagnosticum for RIGA, used to detect diphtheria exotoxin of corynebacteria in a liquid nutrient medium when material from the nose and oropharynx is inoculated into it.

The hemagglutination reaction is used to diagnose both bacterial (typhoid fever, paratyphoid fever, dysentery, brucellosis, plague, cholera, etc.) and viral (influenza, adenoviral infections, measles, etc.) infections. In terms of sensitivity and specificity, RIGA is superior to RA.

Hemagglutination inhibition reaction (HAI)

The hemagglutination inhibition reaction (HAI) is used to titrate antiviral antibodies in blood serum, as well as to establish the type of isolated viral cultures. RTGA can be used to diagnose those viral infections whose pathogens have hemagglutinating properties.

The principle of the method is that serum containing antibodies to a specific type of virus suppresses its hemagglutinating activity and the red blood cells remain non-agglutinated.

Passive hemagglutination inhibition (delay) reaction (RPHA).

Three components are involved in RTPGA: immune serum, antigen (test material) and sensitized erythrocytes.

If the test material contains an antigen that specifically reacts with the antibodies of the immune standard serum, then it binds them, and with the subsequent addition of erythrocytes sensitized with an antigen homologous to the serum, hemagglutination does not occur.

RTPHA is used to detect microbial antigens, for their quantitative determination, and also to control the specificity of RTPGA.

Latex agglutination reaction (RLA)

Latex particles are used as a carrier of antibodies (immunoglobulins). RLA is an express method for diagnosing infectious diseases, taking into account the time required (up to 10 minutes) and the ability to detect antigen in a small volume of test material.

RLA is used to indicate antigens of Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitidis in cerebrospinal fluid, to detect group A streptococci in throat swabs, to diagnose salmonellosis, yersiniosis and other diseases. The sensitivity of the method is 1-10 ng/ml, or 10³ -10⁶ bacterial cells in 1 µl.

Coagglutination reaction (CoA)

The coagglutination reaction (CoA) is based on the ability of protein A of staphylococci to attach specific immunoglobulins. RCA - a method of express diagnostics - serves to identify soluble thermostable antigens in human secretions and in the composition of circulating immune complexes (CIC). Detection of specific antigens in the composition of the CEC requires their preliminary precipitation from blood serum.

Precipitation reaction

In the precipitation reaction (RP), as a result of the interaction of antibodies with highly dispersed soluble antigens (proteins, polysaccharides), complexes are formed with the participation of complement - precipitates. It is a sensitive test used to detect and characterize a variety of antigens and antibodies. The simplest example of high-quality RP is the formation of an opaque precipitation band in a test tube at the boundary of the antigen layering on the immune serum - the ring precipitation reaction. Various types of RP in semi-liquid agar or agarose gels are widely used (double immunodiffusion method, radial immunodiffusion method, immunoelectrophoresis).

Complement fixation reaction (CFR)

The complement fixation reaction (CFR) is based on the phenomenon of hemolysis with the participation of complement, i.e. capable of detecting only complement-fixing antibodies.

RSC is widely used for the diagnosis of many bacterial and viral infections, rickettsial infections, chlamydia, infectious mononucleosis, protozoal infections, helminthiasis. RSC is a complex serological reaction in which two systems are involved: the test (blood serum), represented by the antigen-antibody and complement system, and the hemolytic (sheep red blood cells + hemolytic serum). Hemolytic serum is heat-inactivated rabbit blood serum immunized with sheep erythrocytes. It contains antibodies against sheep red blood cells.

A positive RSC result - the absence of hemolysis - is observed if the test serum contains antibodies homologous to the antigen. In this case, the resulting antigen-antibody complex binds complement, and in the absence of free complement, the addition of the hemolytic system is not accompanied by hemolysis. If there are no antibodies corresponding to the antigen in the serum, the formation of an antigen-antibody complex does not occur, complement remains free and the serum causes hemolysis of red blood cells, i.e. the presence of hemolysis is a negative result of the reaction.

Yushchuk N.D., Vengerov Yu.Ya.

Hemagglutination inhibition reaction

Hemagglutination reaction

Agglutination reaction

Agglutination reaction (RA) is the gluing and precipitation of microbes or other cells under the influence of antibodies in the presence of an electrolyte (isotonic sodium chloride solution). The resulting precipitate is called agglutinate.

For the reaction you need:

1. Antibodies (agglutinins) - are found in the patient’s serum or in immune serum.

2. Antigen - a suspension of live or killed microorganisms, erythrocytes or other cells.

3. Isotonic solution.

The agglutination reaction for serodiagnosis is widely used for typhoid fever, paratyphoid fever (Vidal reaction), brucellosis (Wright reaction), etc. The antibody is the patient's serum, and the antigen is a known microbe.

When microbes or other cells are identified, their suspension serves as an antigen, and a known immune serum serves as an antibody. This reaction is widely used in diagnostics intestinal infections, whooping cough, etc.

In laboratory practice, two hemagglutination reactions (RHA) are used, which differ in the mechanism of action.

First RGA refers to serological. In this reaction, erythrocytes are agglutinated when interacting with the corresponding antibodies (hemagglutinins). The reaction is widely used to determine blood groups.

Second RGA is not serological. In it, gluing of red blood cells is caused not by antibodies, but by special substances formed by viruses. For example, the influenza virus agglutinates the erythrocytes of chickens and guinea pigs, the polio virus agglutinates the erythrocytes of sheep. This reaction makes it possible to judge the presence of a particular virus in the test material.

This is a serological reaction in which specific antiviral antibodies, interacting with the virus (antigen), neutralize it and deprive it of the ability to agglutinate red blood cells, i.e., inhibit the hemagglutination reaction. The high specificity of the hemagglutination inhibition reaction (HITA) allows using it to determine the type and even the type of viruses detected during the HA.

The reaction of indirect (passive) hemagglutination (RIHA) is based on the fact that erythrocytes, if a soluble antigen is adsorbed on their surface, acquire the ability to agglutinate when interacting with antibodies to the adsorbed antigen. The RNGA diagram is shown in Fig. RNGA is widely used in the diagnosis of a number of infections.

Rice. Scheme of the passive hemagglutination reaction (RPHA). A - obtaining an erythrocyte diagnosticum; B - RNGA: 1-erythrocyte: 2 - antigen being studied; 3 - erythrocyte diagnosticum; 4 - antibody to the antigen being studied: 5 - agglutinate.

With the help of RNHA, an unknown antigen can be determined if known antibodies are adsorbed on erythrocytes.

These reactions are called indirect (passive), since they use Ag (or AT) artificially sorbed on the surface of various corpuscular particles.

Indirect or passive hemagglutination reaction (RNGA, RPGA) is one of the most sensitive serological reactions. It is based on the ability of AT to interact with Ag fixed on various red blood cells, which then agglutinate. For greater stability of diagnosticums, red blood cells are formalinized.

Reverse RNGA used to detect Ag in blood serum; For this purpose, not Ag, but AT are fixed on erythrocytes. Reactions of this type are widely used to diagnose infectious diseases, establish pregnancy, detect hypersensitivity to drugs, etc.

Passive hemagglutination inhibition reaction (RTPGA) - further development RNGA; in a sense controls its specificity. Unlike RNGA, includes three components; Ag, AT and Ag (AT) adsorbed on erythrocytes. Initially, Ag reacts with AT (standard antiserum), then erythrocytes sensitized with the same Ag (or AT) are added to the mixture. If, during the interaction of Ag with AT, no free AT (or Ag) remains in the system, then agglutination of the erythrocyte diagnosticum is not observed.